This application addresses broad Challenge Area (04) Clinical Research and Specific Challenge Topic, 04-AI- 101* Develop novel methods and address key questions in mucosal immunology. Influenza viruses are respiratory tract pathogens that infect through the respiratory mucosa and cause significant morbidity and mortality in all age groups. Influenza vaccines, including live attenuated influenza vaccine (LAIV) given via intranasal spay and trivalent inactivated influenza vaccine (TIV) given by systematic intramuscular injection, are effective in protecting children and healthy young adults against influenza virus infection. The mechanisms for the protective efficacy of these different types of vaccines are not completely understood. Although B cell responses are considered a critical component of the influenza mucosal immunity, traditional analyses of B cell immunity after influenza vaccination have been largely limited to studies of serum antibodies, which do not provide adequate or unified markers for the efficacy of LAIV and TIV. The recent finding by us and others of a highly enriched influenza-specific plasmablast pool in the periphery during a narrow window after influenza vaccination provides a unique opportunity to study the phenotypes and functions of vaccine-activated B cells. Taking advantage of this opportunity, we propose the following specific aims: Aim 1. Characterize the trafficking receptor profiles of activated IgA and IgG B cells (plasmablasts) after immunization with LAIV or TIV. An essential component of effective mucosal immunity is targeting of the immune cells to the mucosal site where the infecting pathogen enters and replicates. Lymphocyte trafficking is mediated by the expression of lymphocyte trafficking receptors, which control the multi-step processes of lymphocyte homing, chemotactic navigation, and cell-cell interactions within tissues. We hypothesize that B cells activated by these two vaccines will express distinct trafficking receptors, resulting in a differential ability to home to the respiratory tract. To test this hypothesis, we will collect peripheral blood samples from health adults seven days after LAIV or TIV vaccination and analyze plasmablasts for expression of trafficking receptors using multi-color flow cytometry. Aim 2. Compare the antibodies produced by plasmablasts after LAIV or TIV immunization. The affinity of vaccine-induced antibodies to the vaccine antigens is an important characteristic of antibody response that directly affects the vaccine efficacy. We hypothesize that LAIV- and TIV-induced plasmablasts produce antibodies with distinct antigen binding affinities. Due to the recurring nature of influenza epidemics and the practice of annual influenza vaccination with new vaccine strains, most adults and older children already have a varying number of previous exposures to influenza, resulting in long-life plasma cells and circulating antibodies with variable cross-reactivity to the current influenza vaccine. Therefore, it has been very difficult, if not impossible, to use the conventional serological assays (ELISA, HAI and neutralization assays of serum samples) to specifically identify and characterize the antibodies directly induced by a new vaccination. To test our hypothesis in a way that avoids this interference, we will isolate different subsets of plasmablasts by FACS sorting, based on their expression of phenotypic markers, including trafficking receptors (Aim 1). The sorted plasmablasts will be bulk-cultured ex vivo to collect secreted polyclonal antibodies, which will be tested with ELISA to determine their binding affinity to the influenza vaccine viruses. Aim 3. Profile cytokines produced by activated B cells after LAIV or TIV immunization. Recent findings on the production of cytokines by activated B cells strongly suggest that B cells serve as immune regulators, beyond their conventional role as antibody-producers. We hypothesize that LAIV and TIV immunizations result in B cells that produce distinct panels of cytokines that differentially modulate the immune responses to the two vaccines. To test this hypothesis we will utilize two strategies to compare the cytokine profiles of LAIV- activated versus TIV-activated B cells: 1) Analyze cytokines produced by ex vivo-cultured FACS-sorted plasmablast subsets and 2) Analyze cytokine-encoding mRNA from FACS-sorted plasmablast subsets. Designed to reveal new aspects of influenza B cell immunity, each of these three aims utilizes a novel approach for the phenotypical or functional characterization of B cells directly activated with either LAIV or TIV. These studies will enhance our understanding of the mechanisms for the protective mucosal immunity elicited by the two different influenza vaccines. This is critical for rational design of new influenza vaccines for the annual influenza epidemics and in the face of a potential influenza pandemic. Our experiments are also expected to provide important insights into protective immunity against other medically important viruses that infect the airways and gastrointestinal and vaginal tracts as well as advancing our ability to understand the B cell response to immunization in general. PUBLIC HEALTH RELEVANCE: This study examines how B cells that are recently activated by influenza vaccination are programmed to restrict viral replication. Influenza infection kills over 35,000 people in the United States each year. These studies will provide new information to enhance our ability to improve the influenza vaccines.